We have previously found that 22:6n-3, a highly polyunsaturated n-3 fatty acid, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic neuronal cell death. We have also demonstrated that n-3 fatty acid deficiency or chronic alcohol exposure markedly decreased the PS content specifically in neuronal cells where 22:6n-3 was highly enriched. During dietary n-3 fatty acid deficiency, 22:6n-3 is replaced by docosapentaenoic acid (22:5n-6). In this report period, we examined the effect of 22:6n-3 and 22:5n-6 on the PS content in relation to their ability to prevent neuronal apoptosis. Neuro-2A cells enriched with 22:6n-3 or 22:5n-6 for 48 h at various ratios (1:0, 3:1, 1:1, 1:3 and 0:1 with the final concentration at 25 micromolar) were analyzed for the phospholipid content by reversed phase HPLC/electrospray mass spectrometry or evaluated for the apoptotic cell death induced by serum starvation. The cells enriched with 22:6n-3 or 22:5n-6 alone showed significantly higher levels of the total PS in comparison to non-enriched control, mainly due to the increase of 18:0,22:6n-3 or 18:0,22:5n-6 species, respectively. However, the extent of PS increase due to 22:5n-6 enrichment was less than 70% of the increase observed by 22:6n-3 enrichment. As the proportion of 22:6n-3 with respect to 22:5n-6 increased, a gradual enhancement in the total PS content was observed, indicating that 22:5n-6 is less effective than 22:6n-3 in promoting PS accumulation. Concurrently, the protective effect against apoptotic cell death induced by serum starvation gradually improved with the increasing proportion of 22:6n-3, supporting a strong relationship between PS content and the anti-apoptotic effect of polyunsaturates. In conclusion, 22:5n-6 is not as effective as 22:6n-3 in causing PS accumulation nor in preventing apoptotic cell death, possibly providing a basis for some of the functional deficits associated with n-3 fatty acid deficiency. During this period, we have also extended our investigations into the in vivo effects of n-3 fatty acid and ethanol on neuronal survival and differentiation. Hippocampal cultures were prepared from E18 fetus of rats fed with n-3 deficient diet (15% 18:2n-6 and 0.02% 18:3n-3) or n-3 fatty acid adequate diet (15% 18:2n-6 and 3% 18:3n-3) during pregnancy (E2-E18). Apoptotic cell death induced by overnight trophic factor withdrawal was significantly increased in the deficient group in comparison to the control group. In the deficient fetal hippocampus, 22:6n-3 was replaced by 22:5n-6 and the PS composition was slightly (15%) but significantly decreased as in the adult hippocampus from rats raised on an n-3 fatty acid deficient diet. These results suggest that in vivo PS accumulation promoted by 22:6n-3 plays an important role in preventing apoptotic cell death in the developing hippocampus. When rats were fed with ethanol mixed with an AIN-93G based diet from day 11 of gestation, the PS contents in various brain regions decreased at E18, P0 and P20. E-18 hippocampal cultures from ethanol-fed animals were significantly more sensitive to apoptotic cell death upon withdrawal of trophic factors. The results from the in vitro fatty acid enrichment study using transformed cells as well as in vivo feeding study consistently suggest that PS plays an important role in supporting cell survival. Inappropriate cell death promoted by the depletion of PS either by dietary n-3 fatty acid deficiency or due to prenatal ethanol exposure may have adverse implication in neuronal function. Supplementation of E-18 hippocampal cultures with 1.5 micromolar 22:6n-3 substantially increased neuronal differentiation evaluated by measuring neurite length on neurons positively expressing microtubule associated protein-2 (MAP2). We found that after 6 days in vitro, the DHA supplemented group contained the cell population with longer total neurite length per neuron in comparison to the non-supplemented group. Since the total neurite length is the sum of all neurites derived from a single neuron, increased branching and/or the length of each neurite may account for the difference of the total neurite length per neuron. A similar DHA effect was observed from the culture after 5 days in vitro, but to a lesser degree. Other fatty acids such as oleic (18:1n-9), arachidonic (20:4n-6) or 22:5n-6 did not have any significant effect on neurite extension. These results indicate a unique positive effect of DHA on hippocampal neuronal differentiation, suggesting that DHA may influence neuronal development and function in vivo. In parallel, continuing efforts were made to investigate the protein conformational changes to elucidate the molecular mechanism underlying the effect of 22:6n-3-enriched cell membranes to signal transduction. Using intra-molecular cross linking and mass spectrometric analysis, we were able to establish a method to evaluate the changes in three dimensional structure of proteins. This approach is currently applied to characterize the rhodopsin conformational changes due to visual signal transduction.